Eribulin inhibits growth of cutaneous squamous cell carcinoma cell lines and a novel patient-derived xenograft

Advanced cutaneous squamous cell carcinoma (cSCC) is treated with chemotherapy and/or radiotherapy, but these typically fail to achieve satisfactory clinical outcomes. There have been no preclinical studies to evaluate the effectiveness of eribulin against cSCC. Here, we examine the effects of eribulin using cSCC cell lines and a novel cSCC patient-derived xenograft (PDX) model. In the cSCC cell lines (A431 and DJM-1 cells), eribulin was found to inhibit tumor cell proliferation in vitro as assessed by cell ATP levels. DNA content analysis by fluorescence-activated cell sorting (FACS) showed that eribulin induced G2/M cell cycle arrest and apoptosis. In xenograft models of cSCC cell lines, the administration of eribulin suppressed tumor growth in vivo. We also developed a cSCC patient-derived xenograft (PDX) which reproduces the histological and genetic characteristics of a primary tumor. Pathogenic mutations in TP53 and ARID2 were detected in the patient’s metastatic tumor and in the PDX tumor. The cSCC-PDX responded well to the administration of eribulin and cisplatin. In conclusion, the present study shows the promising antineoplastic effects of eribulin in cSCC. Also, we established a novel cSCC-PDX model that preserves the patient’s tumor. This PDX could assist researchers who are exploring innovative therapies for cSCC.


Eribulin induces cell cycle arrest and cell death in cSCC cell lines. Since the cSCC cells (A431 and
DJM-1) showed high sensitivity to eribulin in vitro, DNA content analysis using flow cytometry was performed to investigate the effect of eribulin on cell cycle progression. At 24 h after the administration of eribulin, the subgroups of A431 and DJM-1 cells that were in the G2/M and sub-G0/G1 phases were increased. Conversely, the subgroups of A431 and DJM-1 cells that were observed in the G0/G1 phase were significantly decreased. NHDF cells presented no significant impact on cell cycle phases (Fig. 2). Furthermore, an increased ratio of early apoptosis was found in A431 cells, and late apoptosis in DJM-1 cells (Supplemental Fig. S1). This result revealed that eribulin induces apoptosis in A431 cells and induces cell death in DJM-1 cells. This finding indicates that the eribulin induced G2/M mitotic block and cell death, as previously reported 6,18,19 .

Administration of eribulin inhibits tumor growth in vivo.
To extend the studies and elucidate the effect of eribulin in vivo, we subcutaneously xenografted cSCC cells (A431 and DJM-1) into immunodeficient nude mice. Compatible with in vitro experimental evidence, both the A431 and DJM-1 xenograft tumors showed reduced growth curves under eribulin treatment (1.5 mg/kg injected intravenously once a week). The weights of extracted tumors in the eribulin-treated group were significantly lower than those in the control group ( Fig. 3A and B). Taken together, the above data reveal that the systemic administration of eribulin suppresses the tumor growth of cSCC in vivo.
Establishment of a cSCC-PDX mouse model harboring TP53 and ARID2 mutations. Few available cSCC-PDX models have been reported 20 ; thus, we tried to develop a mouse model for assessing the efficacy of eribulin (Fig. 4) 21 . Surgically excised tissue was transplanted into the flanks of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. The transplanted cSCC tumor arose from the host and enlarged into a subcutaneous indurated nodule more than 10 mm in diameter over a period of 4 months (generation 0: G0).

Figure 1.
Eribulin suppresses tumor growth in cSCC cell lines in vitro. The sensitivity to eribulin of two cSCC cells (A431 and DJM-1) and of normal human dermal fibroblasts (NHDFs) was determined by ATP assay. 2000 cells per well were seeded in 96-well plates for 24 h. Following that, the cells were treated with eribulin at various concentrations (0.001, 0.1, 0.25, 0.5, 1, 5, 10 nM). After 72 h, cell viability was assessed using CellTiter-Glo ® 2.0 assay (Promega). The x-axis indicates the log value of eribulin concentration; the y-axis indicates the relative cell viability (normalized by no treatment). Data are presented as mean ± SD from triplicate experiments. www.nature.com/scientificreports/ Once the tumor volume exceeded 1000 mm 3 , the cSCC-PDX tumors were transplanted into the next generation of NOD/SCID mice. The patient specimen and xenograft tissue were histologically reviewed by hematoxylin and eosin (HE) staining. The cSCC-PDX tissue manifested identical morphology to that of the patient tissue (primary tumor and lymph node metastasis) (Fig. 4A). Also, we established primary culture cells from the third generation of cSCC-PDX tumors, in which the cultured cells were epithelioid (Fig. 4A). To determine whether the patient's tissues and the cSCC-PDX tumors had consistent genetic similarities, we conducted gene mutation analysis of cancer-associated genes (Supplemental Table S1). Several pathogenic gene mutations, including TP53 R175Pfs*2, ARID2 T1167Lfs*6, BRCA1 S1563C, and BRCA1 E1562Q, were observed to be shared by the patient's cSCC tumor and the cSCC-PDX tumors. The xenograft passage had good concordance with the corresponding tumor tissue of the patient (Supplemental Table S2). In the patient's lymph node, the variant allele frequency (VAF) for TP53 R175Pfs*2 and ARID2 T1167Lfs*6 was 97.2% and 58.3%, respectively. As a result of normal allele loss and the ratio of normal cells diminished in the cSCC-PDX (G1) tumor, the VAF of both mutations was raised to just under 100%. Additionally, there were many more genetic alternations in xenografts due to mutations of several tumor suppressors, including ARID2 22 .
Eribulin suppresses tumor growth in the novel cSCC-PDX mouse model. To date, no preclinical studies on cSCC treated with eribulin have been published. We conducted treatment experiments to study whether the novel cSCC-PDX responds to chemotherapy (Fig. 5) [23][24][25] . Regarding cytotoxic chemotherapies, the xenografted model responded well to cisplatin, which has been reported to be the standard medication 24,25 . Furthermore, we administrated eribulin monotherapy, which has proven effective as a second-line treatment for metastatic breast cancer 26 . As observed in other preclinical models 15 , eribulin administration (1.5 mg/kg/ week) successfully suppressed tumor growth in the cSCC-PDX, and no progression was observed for 2 weeks. week and were estimated based on the formula: (long axis × short axis 2 )/2. The tumors were extracted and measured at 28 days after treatment initiation. The results are shown as mean ± SD. **P < 0.01, ***P < 0.001. www.nature.com/scientificreports/ The eribulin-treated groups showed effects as great as those of the cisplatin-treated group. The data of treatment experiments were additionally validated by Ki-67 staining (Fig. 6). In immunohistochemical analysis, the Ki-67 index score was significantly lower in the eribulin-treated cSCC-PDX tumors than in the control tumors.

Discussion
Cytotoxic agents for advanced cSCC include platinum-based drugs (cisplatin or carboplatin), 5-fluorouracil, bleomycin, methotrexate, doxorubicin, taxanes, capecitabine, and gemcitabine. These have all proven to have insufficient clinical efficacy, and patients have shown several adverse effects [27][28][29] . There have been no preclinical studies on the effectiveness of eribulin against cSCC. In this study, we presented data that demonstrate the significant anti-tumor effects of eribulin against cSCC in vitro and in vivo, suggesting that eribulin could be a novel candidate drug for cSCC treatment.
Previous studies revealed that eribulin suppresses the tumor growth of certain human cancers, including breast, lung, and ovarian cancers, in vitro (IC 50 = 0.09 -9.5 nM) 30,31 . In our study, cSCC cell lines (A431, DJM-1) showed relatively high sensitivity (IC 50 = 0.20 and 0.21 nM) to eribulin, which we have not seen reported before. In cSCC cell lines with a high proliferative rate, a higher proportion of cells abnormally enter the G2/M phase of the cell cycle and cause an irreversible mitotic block. That is why cSCC cell lines are sensitive to eribulin. Even though the growth of cSCC cell lines was inhibited at the sub-nM level of drug administration, the eribulin showed no cytotoxic effects against normal human dermal fibroblasts (NHDFs) up to 1 nM, suggesting that eribulin has high potency with a wide effective range (at sub-nM concentration) 31 . Reportedly, when eribulin was administered to the xenografted mice at a dose of 1 mg/kg, the peak blood concentration exceeded 100 nM, followed by a steady low-concentration phase at around 10 nM for 1 week in xenograft tumors 32 . The present The patient's primary skin lesion was located on the ring finger of the right hand (upper left). Lymph node metastasis was observed 2 years after the first surgery (upper middle). The extracted tumor from the lymph node metastasis was transplanted into NOD/SCID mice (G0, upper right). The enlarged xenografted tumors were transplanted to further generations for primary cell cultures (B) and for treatment study (C). In HE staining and bright-field images, the cSCC-PDX tissue exhibits similar morphology to that of the patient's tissue (metastatic lymph node). (C) In treatment experiments, tumor-bearing NOD/SCID mice were randomized into a control group, a cisplatin group (5 mg/ kg/week), and an eribulin group (1.5 mg/kg/day). www.nature.com/scientificreports/ study showed the IC 50 of eribulin in cSCC cell lines to be at the sub-nM level, suggesting that eribulin has a great anticancer activity. Eribulin blocks microtubule assembly by attaching to a distinctive binding site on tubulin that differs from the sites for other known categories of tubulin-directed drugs 33 . This unique mechanism may explain why eribulin has non-cross-resistant anti-neoplastic effects after the cessation of taxane treatment 34,35 .
Regarding inhibiting mitotic spindle alignment, eribulin administration results in cell cycle arrest at the G2/M phase, leading to cell apoptosis 33 . This phenomenon was also observed in our experiments using flow cytometry analysis and immunohistochemistry.
In the present study, we have reported a novel cSCC-PDX mouse model harboring TP53 R175Pfs*2 and ARID2 T1167Lfs*6 truncating mutations. The mutations could not only result in loss of function (LOF), but could also facilitate tumor progression 36,37 . Concomitant TP53 and ARID2 mutation have been reported in non-small cell lung carcinoma 38 , oral SCC 39 , and colorectal cancer 40 . Concerning clinical research on TP53 mutations in cSCC, the frequency of such mutations is around 50% in primary SCC tumors, but is almost 95% in metastatic tumors [41][42][43] . TP53 controls both G1/S and G2/M cell cycle checkpoints 44 . In contrast, the incidence rate of ARID2 genetic alteration in cSCC is unknown and has not been investigated, but it is reportedly 6% for oral SCC tumors 45 . ARID2 performs a tumor suppressor function in TP53-mutated oral SCC in vitro, and ARID2 knockdown accelerates tumor growth in vivo 36 . The inhibition of ARID2 expression enhances G1/S transition related to the upregulation of cyclin D1, cyclin E1, and CDK4 and to the phosphorylation of the retinoblastoma protein (Rb) in hepatocellular carcinoma (HCC) 46 . In several studies, ARID2 mutations and/or lower protein expression of ARID2 have been reported to correspond to poor patient prognosis in hepatocellular carcinoma, intrahepatic cholangiocarcinoma, breast cancer, oral SCC, and lung cancer [47][48][49][50][51] . Based on our data, eribulin demonstrated high effectiveness against a cSCC-PDX tumor harboring TP53 and ARID2 mutations. Without these two tumor suppressor genes, the surveillance mechanisms at the G1/S and G2/M checkpoints fail to regulate cell cycle, leading to abnormal cell growth and tumorigenesis. The oncogenic effects of these tumor suppressor genes include the initiation of DNA synthesis and enhanced cell growth, invasion, and metastasis. After the administration of eribulin, the irreversible mitotic blockade induced by eribulin leads to cancer cell apoptosis (Fig. 7).
Our results indicate that eribulin induces irreversible mitotic blockade and apoptosis, leading to tumor regression in cSCC cell lines (A431 and DJM-1) as well as in a cSCC-PDX. Regarding A431 cells, there are no TP53 or ARID2 mutations; instead, the A431 cells have the amplification of EGFR, CCND1, and CCND3 and the deletion of CDKN2A. The DJM-1 cells had no TP53 or ARID2 mutations but did have gain of function in FGFR2 and FGFR4, the amplification of MYC, and the deletion of CDKN2A 52 . These cSCCs and the PDX share similarities in cell-cycle dysregulation and contribute to tumorigenesis 53 . Our data suggest that eribulin could have excellent anti-neoplastic effectiveness against cSCC cells regardless of their genetic backgrounds.
In the treatment experiment, we used cisplatin or eribulin only as a single agent. In the cisplatin group, the mice did not tolerate the drug well, becoming weak and losing body weight. In the eribulin group, the mice seemed healthy for the duration of the experiment. We may consider using eribulin in combination with lowdose cisplatin or with EGFR inhibitors as multidrug regimens in the future.
Traditional patient-derived xenograft (PDX) mouse models lack normal immunity. They cannot be used to evaluate the efficacy of anti-PD-1/PD-L1 therapies. In the past few years, some researchers have developed new mouse models where human PDX grows in the presence of a human immune system 54,55 . We may utilize this concept to establish new models for assessing the effectiveness of PD-1/PD-L1 inhibitors in combination with eribulin at a future time. www.nature.com/scientificreports/ In conclusion, the present study reveals the promising antineoplastic effects of eribulin. Also, we established a novel cSCC-PDX mouse model that preserves the patient's tumors histologically and genetically. This mouse model could help researchers who are exploring innovative cSCC therapies.

Material and methods
Cell lines and cell culture. The human cSCC A431 cell line 56 and normal human primary dermal fibroblasts (NHDFs) were purchased from the American Type Culture Collection (ATCC). The human cSCC DJM-1 cells were separated from human cSCC 57 . All of the cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich, Germany). These cells were maintained in a humidified cell incubator containing 5% CO 2 with temperature set at 37 °C. Each cell line was confirmed by short tandem repeat profiling (Promega, WI, USA) in August 2018 and was utilized in less than 6 months of unceasing passage. All cells were verified for the absence of Mycoplasma contamination (Venor ® GeM Classic, Minerva Biolabs, NJ, USA).
Cell viability assays using ATP measurement. Cells were seeded in 96-well solid white plates at a density of 2.0 × 10 3 cells per well in 80 μL of complete medium and were cultured for 24 h. The cells were treated with various concentrations of eribulin (0.001, 0.1, 0.25, 0.5, 1, 5, 10 nM) for 3 days. An ATP assay was performed according to the manufacturer's protocol (CellTiter-Glo ® 2.0 assay, Promega). 50 μL of reagent solution was added to each well, and the plates were kept in the dark for 15 min. The absorbance of luminescence was read at 578 nm in a luminometer (SpectraMax ® Paradigm ® , Molecular Devices, CA, USA) 58 . In vivo experiment on the cSCC-CDX mouse model. The cell line-derived xenograft (CDX) experiments were conducted according to our previous publication 60 . BALB/cAJcl-nu/nu mice (5 weeks old, female) were purchased from CLEA Japan, Inc. The mice in the study were fed with sterile distilled water and standard chow ad libitum. They were held in a fixed-temperature (22-25 °C) environment with a 12-h light/dark cycle under specific pathogen-free conditions. The mice were obtained and housed in accordance with the Animal Resource Program guidelines. 5.0 × 10 6 cells were suspended in 200 μL of PBS and were injected subcutaneously into bilateral flanks. Tumor size was recorded twice per week. For the eribulin treatment group (n = 4, 1.5 mg/kg, Halaven ® , Eisai, Tokyo, Japan), when the tumors reached 8 mm in diameter (usually 5 to 10 days after xenotransplantation), eribulin was injected into the tail vein (30 μg in 150 μL PBS, equivalent to 1.5 mg/kg) and was re-injected every week. The control xenograft tumors (n = 3) were treated with 150 μL PBS. Tumor volume was calculated by an approximation formula: ((1/2) × (major axis) × (minor axis) 2 ) 59 . After 4 weeks of treatment, the tumors were extracted and the weights were recorded.
Establishing the cSCC-PDX. cSCC tissues were obtained from the lymph node metastasis of a 69-yearold Japanese male. He had been a farmer who had habitually smoked cigarettes and drunk alcohol. The patient's primary skin lesion was located on the finger, and he underwent wide excision with amputation for treatment. The pathological report revealed focal positivity upon microscopic examination. The enlargement of ipsilateral axillary lymph nodes was observed 2 years after surgery. The patient died soon from cancer cachexia and sepsis. The resected metastatic lymph node was divided into two sections: one was for transplantation; the other was fixed in formalin and embedded into paraffin for pathological diagnosis. A 10-mm specimen of metastatic tissue from the cSCC patient was subcutaneously transplanted with Matrigel (BD Biosciences) into the bilateral flanks of a 5-week-old female NOD/ShiJic-scidJcl mouse (CLEA Japan, Tokyo, Japan). The tumor-transplanted mouse was checked twice per week and the tumor was measured by caliper. When the tumor volume reached 1000 mm 3 , the cSCC-PDX tumors were propagated to the next generation of mice. In the first two consecutive mouse-to-mouse passages, the cSCC-PDX tumors were divided into three sections: one was cut into pieces (less than 5 mm in diameter) for transplantation, another was frozen at − 80 °C for DNA extraction, and the third was fixed with 10% formalin and then embedded in paraffin for pathological analysis. Treatment experiments were performed on the first to third generations of PDX mice. Extra amounts of fresh tumor pieces were frozen in CryoStor ® CS10 (BioLife Solutions, WA, USA) and stored at − 80 °C 61  www.nature.com/scientificreports/ in the tumor-bearing NOD/ShiJic-scidJcl mice was randomly assigned, and a treatment protocol initiated. Each treatment group had a minimum of n = 3 mice per condition. The control (n = 3) mice were intravenously injected with 150 μL of PBS once per week. In the cisplatin group, cisplatin (n = 4, 5 mg/kg, FUJIFILM Wako, Tokyo, Japan) was administered intraperitoneally once per week as described by Lu et al. 62 . Eribulin (n = 4) was administered intravenously once per week 63 . The tumor volume was measured twice per week by caliper, and tumor weights were measured at 42 days after treatment initiation. Tumor volume and weight were recorded in a blinded manner.
Histopathological analyses. Formalin-fixed, paraffin-embedded tissue sections from the patient's tumors and the xenografted tumors were cut into 4-μm sections. Hematoxylin and eosin (HE) staining and immunohistochemistry for Ki-67 (Abcam, Camb, UK) were performed to compare the histopathology of the metastatic lymph nodes and the xenografts before versus after the treatments. DAB chromogen was applied to yield a brown color 64 . For nuclear Ki-67 expression, the percentage of positive cells among at least 100 cancer cells from three randomly selected fields of vision under high magnification (× 400) was calculated.
Gene mutation analysis. Genomic DNA was extracted from the patient's blood and from each tissue (patient's lymph node and PDX) using the DNA Mini Kit (QIAGEN, Germany). The concentration and purity of DNA samples were measured using a NanoDrop 2000c Spectrophotometer (Thermo Fisher, MA, USA). DNA fragment integrity was confirmed by electrophoresis using 1% agarose gel. The concentrations of DNA samples were normalized to 20 ng/μL, and the samples were stored at − 30 °C until use. Genomic testing was performed by the genomic unit of the Keio Cancer Center in Tokyo, Japan. The quality of the DNA was first determined based on the DNA integrity number (DIN) score, which was calculated using the Agilent 2000 TapeStation system (Agilent Technologies, CA, USA), and then the targeted amplicon exome sequencing for 160 cancer-related genes was performed using the MiSeq sequencing platform (Illumina, CA, USA). The 160 cancer-related genes in the comprehensive cancer panel are listed in Supplemental Table S1. The minimum amount of DNA was 50 ng, and the minimum quality for DNA was that with a DIN score over 3.1. The sequencing data were analyzed using an original bioinformatics pipeline: GenomeJack (Mitsubishi Space Software, Tokyo, Japan).
cSCC-PDX-derived primary cell culture. The establishment of primary culture cells was conducted as previously reported 65 . Tumor tissue from the cSCC-PDX mouse (third generation) was minced and washed with PBS repeatedly. The minced tissue was directly plated onto dishes in a medium of DMEM containing 10% FBS.

Statistical analyses.
Quantitative data are shown as mean ± standard deviation (SD). To evaluate the statistical significance of the treatment groups, the Student's t test was used to estimate the statistical significance between each category. At least three independent experiments were carried out for statistical comparison. The statistical tests were two tailed, and a P-value < 0.05 was considered statistically significant.
Ethics approval and consent to participate. The study protocol complied with the Declaration of Helsinki and was approved by the institutional review board of Hokkaido University Hospital (IRB approval number: 018-0322). The patient provided written informed consent prior to enrollment. The animal study protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Hokkaido University, and all experiments were performed in accordance with the regulations of IACUC of Hokkaido University (approval numbers 22-0037). All methods are reported in accordance with ARRIVE guidelines (https:// arriv eguid elines. org) for the reporting of animal experiments under ethics approval and consent to participate.

Data availability
The datasets generated and analysed during the current study are available from the following DDBJ Sequence Read Archive (DRA), Accession Number: DRR438367-DRR438369. www.nature.com/scientificreports/